This year we recruited, screened and evaluated 85 new participants into the NIAAA Screening protocol #14-AA-0181. We enrolled 115 participants into 5 protocols (14-AA-0144, 14-AA-0192, 15-AA-0031, 15-AA-0186 and 16-AA-0059). The circadian rhythm protocol #14-AA-0187 is on hold due to the pharmacy closure and research on radiotracers is on hold pending completion of the radiotracer laboratory. 1. Effects of Drugs in Human Brain - Effects of Alcohol on Neuroinflammation: There is preclinical evidence that chronic alcohol leads to brain neuroinflammation, which may contribute to cognitive impairment. To assess if in the human brain alcoholism is associated with neuroinflammation we compared the binding of 11CPBR-28, a TSPO ligand that serves as marker for microglial activation, using PET in 15 participants with an alcohol use disorder (AUD) and 15 healthy controls (HC). Contrary to our hypothesis, AUD had decreased 11CPBR-28 binding in brain (standardized uptake values or SUV) compared to HC. SUV correlated negatively with life-time drinking, yet not with brain glucose metabolism, which was also reduced in AUD compared to HC. Interestingly binding of PBR-28 was inversely associated with levels of cholesterol in plasma. Parallel studies in rats exposed chronically to alcohol showed no differences in 11CPBR-28 binding in brain. The mechanism underlying the reduction of 11CPBR-28 binding in the brain of alcoholics is unclear but might reflect changes in binding affinity to TSPO or competition for binding with an endogenous ligand (i.e. cholesterol). 2. Dopamine and Addiction - Subcortical Hyperconnectivity in Cannabis Dependence: Cannabis use has been associated with higher risk of schizophrenia, though the mechanism underlying this are poorly understood. Inasmuch as increased DA signaling in midbrain-striatal circuits is associated with schizophrenia we hypothesized that regular cannabis use would be associated with increased functional connectivity in these circuits. To test this hypothesis, we examined resting brain activity of subcortical regions in 441 young adults, including 30 cannabis abusers (CA) from the Human Connectome Project (HCP) and computed their local functional connectivity density (lFCD). As hypothesized, CA showed markedly increased lFCD relative to controls in the ventral striatum and midbrain but also in lateral thalamus an effect that was most prominent in the individuals who began cannabis use earliest in life. CA reported higher levels of negative emotionality, which was associated with higher subcortical lFCD. These findings suggest that chronic cannabis abuse is associated with changes in dopaminergic nuclei implicated in psychosis and involved with habits and reward processing. Striatal DA D2/D3 Receptor Availability in Smokers: To assess how tobacco smoking status affects DA D2/D3 (D2R) receptor availability and methylphenidate (MP) induced DA release, we analyzed D2R availability measures of 8 current smokers, 10 ex-smokers, and 18 non-smokers who were scanned with PET and 11Craclopride, after an injection of placebo or 0.5 mg/kg iv MP. There was a significant effect of smoking status on striatal D2R availability; with current smokers showing lower D2R compared to non-smokers and to ex-smokers. D2R did not differ between non- and ex-smokers. The effect of smoking status on MP-induced DA release tended to be lower in smokers (p<0.09). For behavioral measures, current smokers showed significantly higher aggression scores compared to non-smokers and ex-smokers. With abstinence ex-smokers may recover from low striatal D2R availability and from increased behavioral aggression. Association of Genetic Ancestry with Striatal DA D2/D3 Receptors: Despite ethnic differences in frequencies of variants in genes associated with D2R availability, no study to date has investigated the relationship between genetic ancestry and striatal D2R. Here, we show that ancestry markers significantly predict dorsal striatal D2R in 117 healthy ethnically diverse residents using PET with 11Craclopride (p<0.0001), while correcting for age, sex, BMI, education, smoking status, and estimated socioeconomic status (ZIP codes). Effects of ethnicity on D2R were not driven by variation in dopaminergic candidate genes. Instead, candidate gene associations with striatal D2R were diminished when correcting for ancestry. Thus, future studies on D2R genes should covary for genetic ancestry or study homogeneous populations. Ancestry studies should also control for socioeconomic differences between ethnic groups since this influence D2R. 3. Methodological studies - Functional connectivity density has neurovascular origin: To assess the relationship between task-induced BOLD activation and deactivation and the changes in functional connectivity density (FCD) we analyzed brain fMRI images from 426 healthy adults at rest and for 7 tasks (working memory (WM), relational processing, reward (gambling), motor function, social cognition, language and emotion) from the HCP. Voxelwise correlations were used to assess the association between functional connectivity (FCD) and BOLD signals. Overall, the main task-induced BOLD activation showed task-related increases in FCD (PFWE < 0.001). Regions with weak BOLD activation and deactivation had task-related decreases in FCD (PFWE < 0.001) that were not associated with BOLD across subjects. Task-related FCD increases and decreases were more pronounced for cognitive and motor than gambling and emotion tasks. The robust associations between BOLD and FCD demonstrates the neurovascular origin of FCD. 4. Glymphatic system- PET Measurement of Beta Amyloid Clearance During Sleep: The brains glymphatic system is implicated in clearance of amyloid-beta (A) and is more active during sleep than wakefulness. We used PET in conjunction with 18Fflorbetaben (FBB) to measure A accumulation in brain after one night of sleep deprivation (SD) and after one night of rested wakefulness (RW) in 20 healthy participants (10 females) in whom we also measured brain glucose metabolism (marker of brain function) during RW. We hypothesized that if the glymphatic system was operational in the human brain then SD would result in amyloid beta accumulation. We found significant increases in A after one night of SD in the hippocampus, which shows amyloid plaque accumulation and decreased glucose metabolism early in Alzheimers disease (AD). Lower reported sleep hours correlated with more A in hippocampus and precuneus where it was negatively associated with glucose metabolism. Our results are consistent with a role of the glymphatic system in clearing A in the human brain and provide evidence that increases in A with worse sleep have functional consequences, as supported by concurrent decreases in metabolism and highlight the relevance of sleep as a target for preventing AD. MRI Measures During Sleep and Wakefulness: Preclinical findings showing increases in the extracellular space associated with glymphatic clearance during sleep led us to hypothesize that if a similar process occurred in the human brain this would be associated with increases in the apparent diffusion of water (ADC) during sleep compared to waking. To test this hypothesis, we tested 20 healthy participants using MRI while awake and while they slept in the MRI scanner. Preliminary analyses revealed a significant increase in total ADC during sleep in the temporal pole and cerebellum and a significant increase in water content in gray matter (MRS) as well as a trend of an increase in the volume of the brain. Our results are consistent with an increase in extracellular space during sleep induced glymphatic clearance.